WO1998038197A1

WO1998038197A1 - Protected aminosugars

Info

Publication number: WO1998038197A1
Application number: PCT/AU1998/000131
Authority: WO
Inventors: Istvan Toth; Gyula Dekany; Barry Kellam
Original assignee: Alchemia Pty. Ltd.
Priority date: 1997-02-28
Filing date: 1998-02-27
Publication date: 1998-09-03
Also published as: EP0977763A1; US6462183B1; HUP0000819A3; EP0977763A4; JP2001513089A; HUP0000819A2; AUPO536797A0; CA2282789A1; CN1248976A

Abstract

The invention provides amine-protecting groups for use in solution phase or solid-phase oligosaccharide synthesis, in which a 2-substituted 1,3-dioxo compound is used to protect one or more primary amine groups of an aminosugar or glycosylamine. The invention provides reagents, reagent kits, and methods for solution phase, solid-phase oligosaccharide synthesis.

Description

PROTECTED AMINOSUGARS

This invention relates to methods for synthesis of oligosaccharides , especially those oligosaccharides which comprise amino sugar residues. In particular the invention relates to methods for solution phase, solid phase or combinatorial synthesis of oligosaccharides.

BACKGROUND OF THE INVENTION Aminosugars are important constituents of various glycoconjugates (Schmidt and Kinzy, 1994). Examples include peptidoglycans, mucopolysaccharides, glycopeptides and proteins, oligosaccharides of human milk, and blood group determinants . They are often also encountered in bacterial and tumour-associated carbohydrate antigens, predominantly in the N-acetylated form or N-acylated with an aspartic acid residue (Toyokuni and Singhal, 1995) . It is therefore evident that these biological glycoconjugates are of immense interest to the medicinal chemist, and therefore that there is a great need in the art to be able to synthesise these compounds in a facile and cost- effective manner.

Oligosaccharide synthesis using aminosugars requires the presence of a suitable amino protecting group. A number of protecting groups have been proposed, but so far all of the agents which are available suffer from serious disadvantages. For example, glycosylation with donors derived from 2-N-acetyl protected aminosugars proceeds via neighbouring group participation; however, formation of the relatively stable oxazoline intermediate dramatically reduces the overall speed and yield of the reaction (Zurabyan et al , 1994) . Therefore, various 2 -deoxy-2-aminosugar donors, displaying the neighbouring group activity described, but lacking the ability to form stable oxazolines, have been developed; the most widely used of these are the phthalimido protected monomers (Sasaki et al , 1978) . The phthalimide group participates strongly during glycoside formation and gives excellent stereocontrol of the 1, 2- trans-glycoside product (Lemieux et al , 1982), furthermore the aminosugar donors do not form stable orthoamides (Lemieux et al, 1982) and cannot form oxazolines. The major disadvantage of using the phthalimide group lies in the vigorous conditions required for its removal, namely heating with methanolic hydrazine, which often results in partial product decomposition. Strongly basic conditions are also required for the removal of the N-sul fonyl (Griffith and Danishefsky, 1990) and N- haloacetyl protecting groups (Shapiro et al , 1967), resulting in similar problems .

The allyloxycarbonyl (Alloc) protected amino sugar donors display a similar activity to their phthalimide counterparts when employed under Lewis acid- catalysed conditions. However, the Alloc group has the advantage that it can be removed under extremely mild conditions, using tetrakis (triphenylphosphine) palladium in the presence of a mild base (Hayakawa et al , 1986) . The major disadvantage associated with the Alloc group lies in its ability to form a stable oxazolidinone intermediate, which in the presence of unreactive acceptors tends to remain as the major product, and reduces the speed and yield of the reaction (Boullanger et al , 1987). 2,2,2- Trichloroethyl-protected aminosugars contain a strongly participating group that, unlike phthalimide, does not deactivate adjacent hydroxyl groups which may subsequently be required as glycosyl acceptors . They can be removed under relatively mild and selective conditions, using zinc and acetic acid, and do not form oxazoline intermediates during glycosylation. However, this protecting group has the disadvantage that benzyl groups cannot be introduced without premature loss of the protecting group as well (Imoto et al , 1987) . Tetrachlorophthaloyl-protected aminosugar donors have been demonstrated to afford high yields of 1 , 2 - trans- glycosides (Castro-Palomino and Schmidt, 1995), even in the presence of poorly reactive acceptors. Once more, however, the NaBH4-mediated deprotection is the limiting factor for this particular protecting group.

The azide group has received much attention in aminosugar chemistry, since it serves as a masked, non- participating amino functionality, thereby allowing the synthesis of 1, 2-cis-linked 2-amino-2-deoxy glycosides (Palsen, 1982). However the preparation of 2-azido-2-deoxy sugars is protracted, costly, and often dangerous, using either azidonitration (Lemieux and Ratcliffe, 1979), diazo- transfer reactions (Buskas et al , 1994), azidochlorination (Bovin et al , 1986), nitrosation of N-benzyl derivatives (Dasgupta and Garegg, 1989) or reactions of 1,6- anhydrosugars (Tailler et al , 1991 and Paulsen and Stenzel, 1978) .

Other non-participating protecting groups that have been reported are 2 , 4-dinitrophenyl (Kaifu and Osawa, 1977) and p-methoxybenzylimino (Mootoo and Fraser-Reid, 1989) , both of which are complicated to introduce and require harsh deprotection conditions which result in loss of product .

A hydrazine-labile primary amino-protecting group, N-l- (4, 4-dimethyl-2 , 6-dioxocyclohexylidene) ethyl (Dde) , has been reported for protection of lysine side chains during SPPS (Bycroft et al , 1993) . This group was modified for use as a carboxy-protecting group in SPPS when the 2- (3-methylbutyryl ) dimedone analogue of 2-acetyldimedone was condensed with 4-aminobenzylalcohol to afford 4- [N- [1- (4, 4-dimethyl-2 , 6-dioxocyclo-hexylidene) -3- methylbutyl] -amino}benzyl ester (ODmab) (Chan et al , 1995). These two protecting groups were reported to be stable to the Fmoc deprotecting conditions widely used in solid phase peptide synthesis (SPPS) , ie 20% piperidine in dimethylformamide (DMF) . Dde has been widely used in the field of SPPS as an orthogonal amino protecting group to the well established Fmoc/t-Boc methodology (Fields and Noble, 1990) . Until now its use has remained within this area, and therefore its use as a protecting group in the field of carbohydrate chemistry is novel. In particular, the use of Dde or ODMab in oligosaccharide synthesis has not been suggested.

We have now surprisingly found that Dde can be used as a non-participating amino sugar protecting group, which can be introduced and removed in a facile and cost- effective manner. We have shown that the vinylogous amide protection afforded by the Dde type group is achieved by simply refluxing the unprotected amino sugar with the precursor, eg. 2-acetyldimedone in the case of Dde, in anhydrous ethanol . Using a Dde-protected aminosugar, we have performed a variety of chemical modifications upon the protected molecule in order to demonstrate the stability of this vinylogous amide type protection. towards commonly encountered reactions involved in carbohydrate modification.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a compound useful as a reagent for solution and/or solid phase synthesis of sugar-containing compounds, comprising a sugar carrying one or more primary amine groups protected with a 2-substituted-l, 3-dioxo compound of General Formula I or General Formula II :

II

in which R¹ and R² may be the same or different, and is each hydrogen or C₁-₄ alkyl,

R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group , and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl .

Any sugar or oligosaccharide bearing an amino group may be used.

In a preferred embodiment, the invention provides a reagent for solution phase synthesis of sugar-containing compounds, comprising a cyclic 2-substituted-l, 3-dioxo compound of General Formula I or II as defined above, in which R' is as defined above.

The compounds of the invention are suitable for use in methods of solid-phase oligosaccharide synthesis, in which sugar units are covalently linked to a resin. Any suitable linker compound may be used. For example, the covalent linkage to the resin may suitably be provided by a

-CONH-, -0-, -S-, -COO-, -CH=N-, -NHCONH-, -NHCSNH, or -NHNH- grouping, eg. Spacer-CONH-resin, Spacer-O-resin, Spacer-S-resin, Spacer-C0₂-resin, Spacer-CH=N-resin, Spacer-NHCONH-resin, Spacer-NHCSNH-resin, Spacer NHNH- resin. Other possible covalent linking groups will be known to those skilled in the art. It is contemplated that linkers and methods described in our International Patent Application No. PCT/AU97/00544 filed on 26 August 1997, are suitable for use with the compounds of this invention. The entire disclosure of PCT/AU97/00544 is incorporated herein by this cross-reference. These linker systems enable solid phase synthesis of oligosaccharides under mild conditions analogous to those used for SPPS.

The resin may be any resin which swells in water and/or in an organic solvent, and which comprises one of the following substituents : halogen, hydroxy, carboxyl , SH, NH₂, formyl, S0₂NH₂, or NHNH₂, for example methylbenzhydrylamine (MBHA) resin, amino or carboxy tentagel resins, paraaminomethylbenzyl (PAM) resin, or 4-sulphamylbenzyl AM resin. Other suitable resins will be known to those skilled in the art . Thus in a second aspect the invention provides a linker-saccharide complex, comprising a linker group and a saccharide compound comprising a protecting group of general formula I or II as defined above, in which the group R' is as defined above. In a third aspect the invention provides a resin- linker-saccharide support for solid-phase oligosaccharide synthesis, comprising a linker group, a resin, and a starting saccharide compound comprising a protecting group of General Formula I or General Formula II as defined above, in which the group R¹ is as defined above.

Any suitable linker may be used. Again, it is contemplated that linkers and methods described in PCT/AU97/00544 may be used.

In a fourth aspect the invention provides a method of solid-phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups, one or more of which is protected as described above, to a resin-linker-saccharide support as described above. In a fifth aspect the invention provides a method of solution phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a linker-saccharide complex as described above. These methods are particularly useful for combinatorial synthetic applications. The solid phase or solution phase method of the invention may, for example, be used for combinatorial synthesis of aminoglycoside compounds. It will be appreciated that the sequential linkage may be effected either enzymically or by chemical means .

The invention also provides a kit for solid phase synthesis, solution phase synthesis, or combinatorial synthesis of oligosaccharides, comprising a linker- saccharide complex or a resin-linker-saccharide support according to the invention, as described above. The kit may optionally also comprise one or more further reagents such as partially or differentially activated, fully protected saccharides, protecting agents, deprotecting agents, resins and/or solvents suitable for solid phase or combinatorial synthesis. The person skilled in the art will be aware of suitable further reagents. Different types of kit can then be chosen according to the desired use .

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

DETAILED DESCRIPTION OF THE INVENTION

Abbreviations used herein are as follows:

Ac acetyl

Bu butyl

Dde N-l- (4, 4-Dimethyl-2 , 6-dioxocyclohexylidene) -ethyl

DMF N, N' -Dimethylformamide

EtOH Ethanol FAB-MS Fast atom bombardment mass spectrometry

Me Methyl

MeOH Methanol

Nde 1- (4-Nitro-l, 3-dioxoindan-2-ylidene) ethyl

NHNde NH-1- (4-nitro-l, 3-dioxoindan-2-ylidene) ethyl NMR Nuclear magnetic resonance

ODmab 4- {N- [1- (4, 4-dimethyl-2 , 6-dioxocyclo-hexylidene) -

3-methylbutyl] -amino}benzyl alcohol

SPPS solid phase peptide synthesis

TBDMS tert-butyl dimethyl silyl tBu tert-butyl

Trt trityl The invention will now be described in detail by way of reference only to the following non-limiting examples, in which the structures of individual compounds are as summarised in the following tables.

Table 1

0)

C m en

H

C H m en x m m

33 c r- m

I (7)

Table 1 (continued)

(A

C 09 (0 H

H C H m en x m m

H c rn t σ>

Table 2

Table 3

Example 1 Synthesis of Dde protected aminosugars

2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6-diσxo-cyclohex-l - ylidene) ethylamino] -D-glucopyranose (1 )

Sodium (143 mg, 6.21 mmol) was added to abs . methanol (30 ml) and the reaction mixture was stirred for 5 min. D-glucosamine hydrochloride (1.34 g, 6.21 mmol) was added to the resulting clear solution and the reaction mixture was stirred at room temperature for another 5 in. 2- Acetyldimedone (1.69 g, 9.32 mmol) was added and the reaction mixture was stirred under reflux for 5 hours. The reaction mixture was cooled and the product was precipitated^" by ether (200 ml) resulting in 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) -ethylamino] -D- glucopyranose (1) (1.66 g, 77.9%).

Rf 0.37 (MeCN/H₂0 10:0.5);

FAB MS C₁₆H₂₅N0₇ (343.33) m/z (%) 366 [M+Na]⁺ (100), 268 (40) , 246 (32) , 224 (15) .

NMR (D₂0) δ 5.12 (d, H-l _β) , 3.95-3.25 (m, 6H, sugar H) , 2.38, 2.36 (2s, 3H, CH₃ ) , 2.28, 2.27 (2s, 4H, 2 CH₂ ) , 0.85 (s, 6H, 2 CH₃) .

Example 2 Synthesis of Dde-protected O-acylated aminosugars 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex-1 -ylidene ) - ethylamino] -1 , 3 , 4 , 6-tetra-O-acetyl - -D-glucopyranose (2)

A mixture of 2-deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -D-glucopyranose (1.55 g, 4.51 mmol), pyridine (11 ml) and acetic anhydride (20 ml) was stirred at room temperature overnight. The reaction mixture was evaporated, and the product was crystallised from MeOH (10 ml) at -15°C to give 2-Deoxy-2- [1- (4, 4-dimethyl-2, 6- dioxocyclohex-1-ylidene) ethyl-amino] - 1,3,4, 6-tetra-O-acetyl-α-D-glucopyranose (2) (1.95 g, 86%).

R_f 0.35 (Hexane/EtOAc 1:1);

FAB MS C24H33NOH (511.50) m/z (%) 534 [M+Na]+ (20), 512 [M+H]⁺ (100), 452 (72), 338 (75).

NMR (CDCI3) δ 13.70 (d, 1H, NH) , 6.22 (d, 1H, H-l, J_{1 2}=3.66 Hz) , 5.40 (t, 1H, H-3), 5.16 (t, 1H, H-4), 4.36 (dd, 1H, H-6') , 4.25 (m, 1H, H-5) , 4.13 (dd, 1H, H-2 ) , 4.05 (dd, 1H, H-6) , 2.58 (s, 3H, CH₃ ) , 2.35 (s, 4H, 2 CH₂ ) , 2.09, 2.03, 1.97 (3s, 9H, 3 AcO) , 1.00 (s, 6H, 2 CH₃ ) .

Example 3 ^" Synthesis of Dde-protected halogenated aminosugars 2-Deoxy-2- [1- (4, 4 -dimethyl -2 , 6-dioxocyclohex-l-ylidene) - ethylamino] -3,4, 6-tri-O-acetyl- -D-glucopyranosyl bromide (3) A mixture of 2-Deoxy-2- [1- (4 , 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -1,3,4, 6-tetra-O-acetyl- α-D-glucopyranose (100 mg, 0.19 mmol) and HBr in acetic acid (45%) (1.0 ml) was stirred at room temperature for 30 min. The reaction mixture was diluted with cold CH C1₂ (10 ml), washed twice with cold H 0 (30 ml), saturated

NaHCθ3 solution (20 ml) and with H 0 again (20 ml ) . The organic phase was dried over MgSθ4 and evaporated, giving 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) - ethylamino] -3,4, 6-tri-O-acetyl-α-D-glucopyranosyl bromide (3) (95 mg, 91%) .

R_f 0.35 (Hexane/EtOAc 1:1);

FAB MS C₂2H₃₀BrNO9 (532.37) m/z (%) 534 [M+H] + (100), 452 (45) , 441 (42) , 338 (77) .

H NMR (CDC1₃) δ 13.83 (d, 1H, NH) , 6.41 (d, 1H, H-l, J_{1 2}=3.65 Hz), 5.52 (t, 1H, H-3 ) , 5.20 (t, 1H, H-4) , 4.38 (m, 2H, H-6*, H-2), 4.24 (m, 1H, H-5), 4.14 (dd, 1H, H-6), 2.62 (s, 3H, CH3 ) , 2.41 (s, 4H, 2 CH₂ ) , 2.11, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 (s, 6H, 2 CH₃ ) . Example 4 Synthesis of Dde-protected O-alkylated aminosugars Methyl 2 -Deoxy-2~ [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethylamino] -3 , 4 , 6-tri -0- acetyl - -D-glucopyranoside (4 )

2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -3,4, 6-tri-O-acetyl-α-D- glucopyranosyl bromide (60 mg, 0.11 mmol) was dissolved in CH₂C12 (5 ml), cooled to -15°C and silver trifluoro-methanesulphonate (43 mg, 0.16 mmol) in MeOH (1 ml) added. The reaction mixture was stirred overnight, filtered and the filtrate evaporated. The residue was washed with saturated NaHC03 solution, dried over MgSθ4 and evaporated. The residue was purified by chromatography, to give Methyl 2-Deoxy-2- [1- (4 , 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) ethylamino] -

3 , 4, 6-tri-O-acetyl-β-D-glucopyranoside (4) (40 mg, 75%).

R_f 0.35 (Hexane/EtOAc 1:1);

FAB MS C ₃H33NO₁₀ (483.49) m/z (%) 506 [M+Na]⁺ (15), 484 [M+H]⁺ (100) , 442 (8) .

!H NMR (CDC1₃) δ 13.84 (d, 1H, NH) , 5.20 (t, 1H, H-3 ) , 5.09 (t, 1H, H-4), 4.41 (d, 1H, H-l, J_1/2=8.29 Hz), 4.32 (dd, 1H, H-2), 4.14, 3.94 (2m, 2H, H-6), 3.75 (m, 1H, H-5) , 3.48 (s, 3H, OCH₃), 2.57 (s, 3H, CH3 ) , 2.37 (s, 4H, 2 CH₂), 2.09, 2.03, 1.96 (3s, 9H, 3 AcO), 1.02 (s, 6H, 2 CH3 ) , and

Methyl 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethylamino] - 3 , 4 , 6-tri -O-acetyl -α-D-glucopyranoside

R_f 0.33 (Hexane/EtOAc 1:1);

FAB MS C₂₃H33NO₁₀ (483.49) m/z (%) 506 [M+Na]⁺ (13), 484 [M+H]⁺ (100) . !H NMR (CDCI3) δ 13.55 (d, 1H, NH) , 5.40 (t, 1H, H-3 ) , 5.08 (t, 1H, H-4), 4.82 (d, 1H, H-l, J_lf2=3.37 Hz), 4.32 (dd, 1H, H-2), 4.12 (m, 3H, H-6, H-5), 3.53 (s, 3H, OCH3), 2.58 (s, 3H, CH3), 2.41 (s, 4H, 2 CH₂ ) , 2.11, 2.02, 1.94 (3s, 9H,^~ 3 AcO), 1.02 (s, 6H, 2 CH3 ) .

Example 5 Synthesis of Dde-protected aminosugar uronium salts S- [2-Deoxy-2 - [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex-1 -ylidene) - ethylamino] -3 , 4 , 6-tri -0-acetyl - -D-glucopyranosyl ] - isothiouroni um bromide (5)

Thiourea (14 mg, 0.18 mmol) was added to a solution of 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -3,4, 6-tri-O-acetyl-α-D-glucopyranosyl bromide (100 mg, 0.18 mmol) in acetone (0.5 ml). The mixture was refluxed for 15 in then evaporated. The residue was purified by chromatography using CHCl3/MeOH 5:1 as the mobile phase to give S- [2-Deoxy-2- [1- (4, 4-dimethyl- 2 , 6-dioxocyclohex-l-ylidene) ethylamino] -3,4, 6-tri-O-acetyl- β-D-gluco-pyranosyl] isothiouronium bromide (5).

R_f 0.46 (CHCl /MeOH 5:1);

FAB MS C₂₃H34N3θ₉S (608.42) m/z (%) 528 [M-Br] + (20), 452 (100) .

¹H NMR (CDCI3) δ 13.85 (d, 1H, NH) , 5.30 (t, 1H, H-3), 5.12 (t, 1H, H-4), 4.75 (d, 1H, H-l, J_{1 2}=9.43 Hz), 2.62 (s, 3H, CH₃), 2.36 (s, 4H, 2 CH₂ ) , 2.11, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 (s, 6H, 2 CH ) . Example 6 Synthesis of Dde-protected alkylthiolated aminosugars Methyl 2-Deoxy-2 - [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethylamino] -l -thio-3 , 4 , 6- tri-O-acet l-β-D- glucopyranoside (6)

2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -l-thio-3 , 4, 6-tri-O-acetyl-β-D- glucopyranose (72 mg, 0.148 mmol) was dissolved in acetone (0.15 ml) and K₂C0₃ (23 mg) in water (0.15 ml) added. The reaction mixture was stirred under N at room temperature and methyliodide (23 mg, 0.163 mmol) added. After 30 min stirring the reaction mixture was concentrated under reduced pressure. CH₂C1₂ (2ml) was added to the reaction mixture and the layers were separated. The organic phase was washed with water (0.5 ml), dried over MgSθ4 and evaporated. The residue was purified by chromatography using EtOAc/hexane 3:1 to give Methyl 2-Deoxy-2- [1- (4, 4- dimethyl-2 , 6-dioxocyclohex-l-ylidene) ethylamino] -1-thio- 3,4,6-tri- O-acetyl-β-D-glucopyranoside (6) (50 mg, 67%).

Rf 0.41 (EtOAc/hexane 3:1);

FAB MS C23H33NO9S (499.49) m/z (%) 522 [M+Na]⁺ (25), 500 [M+H]⁺ (100), 452 (27), 338 (35).

NMR (CDCI3) δ 13.96 (d, 1H, NH) , 5.22 (t, 1H, H-3), 5.13 (t, 1H, H-4), 4.61 (d, 1H, H-l, J_1/2=9.98 Hz), 4.30 (dd, 1H, H-2), 4.15 (m, 2H, H-6', H-5) , 2.60 (s, 3H, CH3 ) , 2.42 (s, 4H, 2 CH₂), 2.20 (s, 3H, SCH3 ) , 2.09, 2.02, 1.96 (3s, 9H, 3 AcO), 1.03 (s, 6H, 2 CH3 ) .

Example 7 Synthesis of Dde-protected benzylated aminosugars Benzyl 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethylamino] -α-D- glucopyranoside (7)

A solution of Benzyl 2-Acetamido-2-deoxy-α-D- glucopyranoside (4.70 g, 15.11 mmol) in 1 M NaOH solution was refluxed at 120°C for 15 h. The reaction mixture was cooled to room temperature, neutralised with 1 M HC1 solution and concentrated. The residue was dissolved in dry EtOH (50 ml) and filtered. 2-Acetyldimedone (4.11 g, 22.6 mmol) and N,N- diisopropylethylamine (2 ml) were added to the filtrate, and the mixture was refluxed for 2 h. The reaction mixture was evaporated to dryness, and the residue was taken up in EtOAc (50 ml) , washed with 1M KHSO4 solution, brine, and evaporated to give Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) ethylamino] -α- D-glucopyranoside (7) (3.78 g, 58%).

Rf 0.43 (CH₂Cl₂/EtOAc/MeOH 10:7:3);

FAB MS C₂₃H₃₁N0₇ (433.48) m/z (%) 456 [M+Na]+ (45), 434 [M+H]+ (100), 452 (30), 338 (25).-

iH NMR (CDCI3) δ 13.44 (d, 1H, NH) , 7.33 - 7.21 (m, 5H, 5 Ar-H) , 4.80 (d, 1H, H-l, J_{1 2}=3.45 Hz), 4.71, 4.56 (2d, 2H, CH₂Ar) , 2.45 (s, 3H, CH₃ ) , 2.31 (s, 4H, 2 CH₂ ) , 0.99 (s, 6H, 2 CH3) .

Example 8 Synthesis of Dde-protected azido derivative of aminosugars 2-Deoxy-2- [1 - (4, 4 -dimethyl -2 , 6-dioxocyclohex-l -ylidene) - ethylamino] -3 , 4 , 6-tri -0-acetyl - -D-glucopyranosyl azide (8)

A mixture of 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -3,4, 6-tri-O-α-D- glucopyranosyl bromide (100 mg, 0.18 mmol), sodium azide 100 mg, 1.56 mmol) in DMF (5 ml) was stirred at 80°C for

2 hours. The reaction mixture was evaporated, taken up in CH₂C1₂ (10 ml), washed with H₂0 (2 x 2 ml), dried over MgS04 ^and concentrated. The residue was purified by chromatography, using hexane/EtOAc 1:1 as the mobile phase, to give 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -3,4, 6-tri-O-acetyl-β-D-glucopyranosyl azide (8) (65 mg, 70%) . Rf 0.55 (hexane/EtOAc 1:1);

FAB MS C H3₀N O₉ (494.48) m/z (%) 517 [M+Na]⁺ (15), 495 [M+H]⁺ (100)^", 452 (10), 338 (25).

l-H NMR (CDC1₃) δ 13.91 (d, 1H, NH) , 5.19 (t, 1H, H-3), 5.10 (t, 1H, H-4), 4.87 (d, 1H, H-l, J_1/ =8.95 Hz), 4.34 (dd, 1H, H-2), 4.15 (dd, 1H, H-6'), 3.85 (m, 2H, H-5, H-6), 2.59 (s, 3H, CH3 ) , 2.38 (s, 4H, 2 CH₂ ) , 1.02 (s, 6H, 2 CH₃) .

Example 9 Synthesis of Dde-protected thiolated aminosugars 2-Deoxy-2- [1 - (4 , 4 -Dimethyl -2 , 6-dioxocyclohex-l -ylidene) - ethylamino] -l -thio-3 , 4 , 6-tri -O- acetyl - -D-glucopyranose (9)

To S- [2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -3,4, 6-tri-O-acetyl-β-D- glucopyranosyl] isothiouronium bromide (136 mg, 0.22 mmol) a solution of Na S θ5 (43 mg, 0.225 mmol) in water (0.2 ml) and 1, 2-dichloroethane (0.24 ml) was added. The reaction mixture was kept under reflux at 85°C for 20 min. After dilution with CH₂C1₂ (5 ml), the layers were separated, the organic phase was washed with water (3 ml) , dried over MgS04_/ concentrated under reduced pressure, and chromatographed using ether /MeOH 10:1 to give 2-Deoxy-2- [1- (4, 4-dimethyl-2, 6-dioxocyclohex-1-ylidene) ethylamino] -1- thio-3,4, 6-tri-O-acetyl-β-D-glucopyranose (9) (95 mg, 87%).

Rf 0.31 (ether/MeOH 10:1);

FAB MS C₂ H₃₁N0₉S (485.47) m/z (%) 508 [M+Na]⁺ (15), 486 [M+H]⁺ (100), 452 (33), 338 (20).

^XH NMR (CDCI3) δ 13.97 (d, 1H, NH) , 5.32 (t, 1H, H-3 ) , 5.15 (t, 1H, H-4), 4.75 (dd, 1H, H-l, J_1/2=8.29 Hz), 3.85 (m, IH, H- 5), 2.62 (s, 3H, CH₃ ) , 2.38 (s, 4H, 2 CH₂ ) , 2.10, 2.04, 1.96 (3s, 9H, 3 AcO), 1.02 (s, 6H, 2 CH₃ ) .

Example 10 Synthesis of Dde-protected benzylidene derivative of aminosugars

Benzyl 4, 6 -O-Benzylidene-2 -deoxy-2 - [1- (4, 4 -dimethyl -2 , 6- dioxocyclohex-1 -yl idene ) ethylamino] - -D-glucopyranoside (10)

A mixture of benzaldehyde (1 ml), formic acid (1 ml) and Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -α-D-glucopyranoside (433 mg, 1 mmol) was stirred at room temperature for 2 h. The reaction mixture was evaporated to dryness using a high vacuum rotary evaporator. The residue was treated with ether (40 ml) and the suspention filtered. The solid purified by chromatography, using CHCl3-EtOAc 10:4 as the mobile phase, to give Benzyl 4 , 6-0-Benzylidene-2-deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) -ethylamino] - α-D-glucopyranoside (10) (340 mg, 65%) .

Rf0.38 (CHCl3-EtOAc 10:4);

FAB MS C30H35NO7 (521.58) m/z (%) 544 [M+Na]⁺ (10), 522 [M+H]+ (100) , 338 (40) .

!H NMR (CDCI3) δ 13.52 (d, IH, NH) , 7.37 - 7.26 (m, 10H, 10 Ar-H) , 5.56 (s, IH, CH-Ar) , 4,90, 4.60 (2d, 2H, CH -Ar) , 4.79 (d, IH, H-l, J_1/2=3.08 Hz), 4.35 (t, IH, H-4), 4.26 (dd, IH, H-2), 3.98 (m, 2H, H-5, H-3), 3.77 (t, IH, H-6'), 3.63 (t, IH, H-6) , 2.57 (s, 3H, CH3 ) , 2.33 (s, 4H, 2 CH₂) , 1.01 (s, 6H, 2 CH3 ) . Example 11 Synthesis of Dde - protected reducing aminosugars 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex- 1 -ylidene ) - ethylamino] -3 , 4 , 6-tri -O-acetyl - -D-glucopyranose (12) Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -α-D-glucopyranoside (400 mg, 0.92 mmol) was dissolved in pyridine (6 ml) and cooled to 0°C, then acetic anhydride (10 ml) was added dropwise. The solution was stirred at room temperature overnight, then evaporated. The residue was purified by chromatography using EtOAc/hexane 3:1 to give Benzyl 2- Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-1-ylidene) - ethylamino] -3,4, 6-tri-O-acetyl-α-D-glucopyranoside (11) (465 mg, 90%) .

R_f 0.41 (EtOAc/hexane 3:1);

FAB MS C₂₉H₃7NO₁₀ (559.59) m/z (%) 532 [M+Na]⁺ (15), 560 [M+H]⁺ (100), 452 (20), 338 (55).

¹H NMR (CDC1₃) δ 13.66 (d, IH, NH) , 7.43 - 7.32 (m, 5H, 5 Ar-H), 5.45 (t, IH, H-3), 5.07 (t, IH, H-4), 4.93 (d, IH, H-l, J_1/2=3.53 Hz), 4.76, 4.72 (2d, 2H, CH₂-Ar) , 4.29 (dd, IH, H-2), 4.07 (m, 2H, H-6', H-5) , 3.96 (dd, IH, H-6) , 2.52 (s, 3H, CH₃ ) , 2.38 (s, 4H, 2 CH₂ ) , 2.10, 2.00, 1.94 (3s, 9H, 3 AcO), 1.03 (s, 6H, 2 CH3 ) .

Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxo- cyclohex-1-ylidene) ethylamino] -3,4, 6-tri-O-acetyl-α-D- glucopyranoside (11) (100 mg, 0.17 mmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10%) (20 mg) overnight. The suspension was filtered, and the filtrate was evaporated to give 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -3,4, 6-tri-O-acetyl-α-D- glucopyranose (12) (75 mg, 90%) .

R_f 0.44 (CHCl₃/EtOAc 1:1); FAB MS C₂₂H₃₁NO₁₀ (469.47) m/z (%) 492 [M+Na]⁺ (45), 470 [M+H]⁺ (100) , 452 (10) .

!H NMR (CDCI3) δ 13.81 (d, IH, NH) , 5.49 (t, IH, H-3), 5.28 (d, IH, H-l, J_1/2=3.29 Hz), 5.11 (t, IH, H-4) , 4.42 (dd, H, H-2), 4.33 (dd, H, H-6'), 2.59 (s, 3H, CH₃ ) , 2.37 (s, 4H, 2 CH₂), 2.10, 2.03, 1.96 (3s, 9H, 3 AcO), 1.01 (s, 6H, 2 CH₃) .

Example 12 Synthesis of Dde-protected trichloroacetimidate of aminosugars 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex- 1 -ylidene) - ethylamino] -3 , 4 , 6-tri -O-acetyl -α, -D-gl ucopyranosyl trichloroacetimidate (13 )

A mixture of 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -3,4, 6-tri-O-acetyl-α-D- glucopyranose (100 mg, 0.21 mmol) and trichloroacetonitrile in CH₂C1 was cooled to 0°C and 1, 8-diazabicyclo (5.4.0) - undec-7-en (2 mg) added. The reaction mixture was stirred at 0°C for 1.5 h and at room temperature for 2 h. The solution was evaporated, and the residue chromatographed using CHCl3/EtOAc 1:1 as the mobile phase to give 2-deoxy- 2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l-ylidene) ethylamino] - 3,4, 6-tri-O-acetyl-α, β-D-glucopyranosyl trichloroacetimidate (13) (71 mg, 55%).

R_f 0.61 (CHCl /EtOAc 1:1);

FAB MS C₂4H₃₁Cl3N2θ₁₀ (613.88) m/z (%) 635 [M+Na]⁺ (75), 452 (100) .

!H NMR (CDCI3) δ 13.95, 13.72 (2d, IH, NH _β), 8.84,

8.76 (2s, IH, NH_{A β}), 6.48 (d, H-l_α, Ji ₂= ³-^{05 Hz}>' 5.85 (d, H-lβ, J_1/2=8.72 Hz), 5.52 (t, IH, H-3), 5.31 (t,

IH, H-4), 2.65, 2.63 (2s, 3H, CH_3(X/β), 2.31 (2s, 4H, 2 CH_2(X/β) , 2.09, 2.08, 2.05, 2.04, 1.99, 1.97 (6s, 9H,

3 AcO_{tt β}) , 0.99, 0.98 (2s, 6H, 2 CH_{3ct β}) .

Example 13 Synthesis of Dde-protected O-triphenylmethylated aminosugars

Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] - 6 ^' -0-triphenylmethyl -α-D-glucopyranoside (14)

A mixture of Benzyl 2-Deoxy-2- [1- (4 , 4-dimethyl- 2 , 6-dioxocyclohex-l-ylidene) ethylamino] -α-D-glucopyranoside (100 mg, 0.23 mmol), triphenylmethylbromide (149 mg, 0.46 mmol) in DMF/pyridine 1:1 (2 ml) was stirred at 100°C for 15 h. The reaction mixture was evaporated, the residue was taken up in CHCI3 (10 ml), washed with water (3 ml), dried over MgSθ4 and concentrated. The residue was purified by chromatography using CHCl3/MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2, 6- dioxocyclohex-1-ylidene) ethylamino] -6-O-triphenylmethyl-α- D-glucopyranoside (14) (104 mg, 64%) .

R_f 0.55 (CHCl₃/MeOH 10:1);

FAB MS C4₂H ₅Nθ7 (675.68) m/z (%) 698 [M+Na]⁺ (40), 676 [M+H]+ (100) .

¹H NMR (CDCI3) δ 13.49 (d, IH, NH) , 7.49 - 7.23 (m, 20H, 20 Ar-H), 4.87, 4.66( 2d, 2H, CH₂Ar) , 4.83 (d, IH, H-l, J_{1 2}=3.70 Hz), 3.84 (t, IH, H-3), 2.55 (s, 3H, CH3 ) , 2.31 (s, 4H, 2 CH₂), 1.02 (s, 6H, 2 CH3 ) .

Example 14 Synthesis of Dde-protected O-silylated aminosugars Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -6-O-t-butyldimethylsilyl-α-D- glucopyranoside (15)

Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -α-D-glucopyranoside (100 mg, 0.23 mmol) was dissolved in dry pyridine (2 ml), cooled to 0°C and t-butyldimethylsilylchloride (39 mg, 0.26 mmol) added. The reaction mixture was stirred at room temperature overnight. The solution was evaporated, the residue was taken up in CHCI3 (10 ml) , washed with water

(3 ml) , dried over MgΞθ4 and concentrated. The residue was purified by chromatography using CHCl3/MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -6-O-t-butyldimethyl- silyl-α-D-glucopyranoside (15) (77 mg, 61%) .

R_f 0.57 (CHCl₃/MeOH 10:1);

FAB MS C₂9H ₅N0₇Si (547.74) m/z (%) 570 [M+Na] ⁺ (10), 548 [M+H]⁺ (100) .

!H NMR (CDCI3) δ 13.45 (d, IH, NH) , 7.40-7.27 (m, 5H,

5 Ar-H), 4.88, 4.65( 2d, 2H, CH₂Ar) , 4.79 (d, IH, H-l,

J_1/2=3.42 Hz), 2.55 (s, 3H, CH₃ ) , 2.31 (s, 4H, 2 CH₂ ) , 1.02 (s, 6H, 2 CH₃), 0.93 (s, 9H, 3 CH₃ ) , 0.10 (s, 6H, 2 CH₃Si) .

Example 15 Synthesis of partially protected polyaminosugars 2 -Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex-1 -ylidene) - ethylamino] -3 , 4 , 6-tri -0-acetyl - -D-glucopyranosyl amine (16)

2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -3,4, 6-tri-O-acetyl-β-D-glucopyranosyl amine (60 mg, 0.12 mmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10%) (10 mg) overnight. The suspension was filtered, the filtrate was evaporated to give 2-Deoxy-2- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethylamino] -3,4, 6-tri-O-acetyl-β-D-glucopyranosyl amine (16) (45 mg, 80%) .

Rf 0.38 (EtOAc) ; FAB MS C₂ H₃₂N θ9 (468.50) m/z (%) 491 [M+Na]⁺ (100), 469 [M+H]⁺ (25) , 452 (10) .

^XH NMR (CDCI3) δ 13.75 (d, IH, NH) , 2.61 (s, 3H, CH3 ) , 2.35 (s, 4H, 2 CH₂), 2.09, 2.02, 1.98 (3s, 9H, 3 AcO), 1.03 (s, 6H, 2 CH₃) .

Example 16 Synthesis of Dmab-protected sugars 4 - [N- [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohexylidene) -ethyl] - amino] benzyl (1 , 2, 3 , 4 -tetra-0-acetyl - -D-glucopyranose) - uronate (17)

A mixture of 1, 2 , 3 , 4-tetra-O-acetyl-β-D- glucuronic acid (100 mg, 0.27 mmol), 4- [N- [1- (4, 4-dimethyl- 2 , 6-dioxocyclohexylidene) ethyl] amino] benzyl alcohol (79 mg,

0.27 mmol), 1,3- dicyclohexylcarbodiimide (62 mg,

0.30 mmol) in CH₂C1₂ was stirred overnight at room temperature. The reaction mixture was evaporated, the residue was purified by chromatography using CHCl3/EtOAc 10:4 to give 4- [N- [1- (4, 4-dimethyl-2 , 6-dioxocyclohexyl- idene) ethyl] -amino] benzyl (1,2,3, 4-tetra-O-acetyl-β-D- glucopyranose) uronate (17) (92 mg, 53%) .

R_f 0.51 (CHCl₃/EtOAc 10:4);

FAB MS C3₁H ₇N0₁3 (631.61) m/z (%) 654 [M+Na]+ (10), 632 [M+H]⁺ (35) , 270 (100) .

^XH NMR (CDCI3) δ 15.06 (d, IH, NH) , 7.41 (d, 2H, 2 Ar-H), 7.15 (d, 2H, 2 Ar-H), 5.76 (d, IH, H-l, J_1/2=9.08 Hz), 4.22 (d, IH, H-5, J_{1 2}=9-36 Hz), 2.51 (s, 3H, CH₃ ) , 2.37 (s, 4H, 2 CH₂), 2.09, 2.00, 1.86 (3s, 9H, 3 AcO), 1.07 (s, 6H, 2 CH ) . Example 17 Synthesis of Dde- and N-acyl-protected polyaminosugars 2-Acetamido-3 , 4 , 6-tri -O-acetyl -1 , 2 -dideoxy-1 - [ 1 - ( 4 , 4 - dimethyl -2 , 6 -dioxocyclohex- 1 -ylidene ) ethylamino] -β-£>- glucopyranose (19)

2-Acetamido-2-deoxy-3 , 4, 6-tri-O-acetyl-β-D- glucopyranosyl azide (100 mg, 0.26 mmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10%) (10 mg) for 5 h. The suspension was filtered, and the filtrate was evaporated to give 2-Acetamido-2-deoxy-3 , 4, 6-tri-O-acetyl- β-D-glucopyranosyl amine (18) (80 mg, 86%) .

R_f 0.38 (CHCl₃/MeOH 10:1);

FAB MS C₁ H₂₂N₂0₈ (346.34) m/z (%) 347 [M+H] + (100), 330 (25).

¹H NMR (CDC1₃) δ 5.64 (d, IH, NH) , 3.99 (m, IH, H-2), 3.65 (m, IH, H-5), 2.11, 2.04, 2.02, 1.97 (4s, 12H, 3 AcO, AcNH) .

A mixture of 2-Acetamido-2-deoxy-3 , 4, 6-tri-O- acetyl-β-D-glucopyranosyl amine (80 mg, 0.23 mmol) and 2-acetyldimedone (55 mg, 0.30 mmol) in MeOH (5 ml) was refluxed for 5 h. The reaction mixture was evaporated, the residue was purified by chromatography using CHCl3/MeOH 10:0.5 as the mobile phase, to give 2-Acetamido-3 , 4, 6-tri- 0-acetyl-l,2-dideoxy-l- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex- 1-ylidene) ethylamino] -β-D-glucopyranose (19) (70 mg, 60%).

Rf 0.37 (CHCl₃/MeOH 10:0.5);

FAB MS C₂ H34N2θ₁₀ (510.53) m/z (%) 533 [M+Na]⁺ (80), 511 [M+H]⁺ (100) , 330 (25) .

!H NMR (CDCI3) δ 13.60 (d, IH, NH) , 5.81 (d, IH, NH) , 5.45 (t, IH, H-3), 5.31 (m, IH, H-l), 5.05 (t, IH, H-4; 4.21 (dd, IH, H-6'), 4.11 (dd, IH, H-6), 3.92 (m, IH, H-2), 3.82 (m, IH, H-5), 2.58 (s, 3H, CH₃ ) , 2.35 (s, 4H, 2 CH₂ ) , 2.06, 2.04, 2.02, 1.92 (3s, 9H, 2 AcO, AcNH) , 1.01 (s, 6H, 2 CH₃ ) .

Example 18 Synthesis of Dde-protected O-isopropylidene derivative of aminosugars Benzyl 2-Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethylamino] -4 , 6-O-isopropylidene-α-D- glucopyranoside (20)

A mixture of Benzyl 2-Deoxy-2- [1- (4 , 4-dimethyl- 2 , 6-dioxocyclohex-l-ylidene) ethylamino] -α-D-glucopyranoside (100 mg, 0.23 mmol) and (+/-) -10-camphorsulphonic acid (5 mg) in 2,2- dimethoxypropane (10 ml) was refluxed for 2 h. The reaction mixture was evaporated, and the residue was taken up in CH₂C1₂ (10 ml) , washed with saturated NaHCθ3 solution (3 ml) , and concentrated. The residue was purified by chromatography using CH₂Cl₂/MeOH 10:1 as the mobile phase to give Benzyl 2-Deoxy-2- [1- (4 , 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -4, 6-0-isopropylidene-α- D-glucopyranoside (20) (82 mg, 75%) .

Rf 0.44 (CH₂Cl₂/MeOH 10:1);

FAB MS C₂₆H₃₅N0₇ (473.54) m/z (%) 496 [M+Na]⁺ (20), 474 [M+H]⁺ (100) , 382 (15) .

¹H NMR (CDC1₃) δ 13.48 (d, IH, NH) , 7.38 - 7.27 (m, 5H, 5 Ar-H), 4.97, 4.65( 2d, 2H, CH₂Ar) , 4.76 (d, IH, H-l, J_1/2=3.55 Hz), 2.55 (s, 3H, CH3 ) , 2.31 (s, 4H, 2 CH₂ ) , 1.52, 1.30 (2s, 6H, 2 CH₃ ) , 1.00 (s, 6H, 2 CH₃ ) . Example 19 Synthesis of Dde- protected galacto- aminosugars 2 -Deoxy-2- [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex- 1 -ylidene) - ethylamino] -D-galactopyranose (21 ) Sodium (22 mg, 0.95 mmol) was added to abs . methanol (10 ml) and the reaction mixture was stirred for 5 min. D-galactosamine hydrochloride (206 mg, 0.95 mmol) was added to the resulting clear solution, and the reaction mixture was stirred at room temperature for another 5 min. 2-Acetyldimedone (261 mg, 1.43 mmol) was added and the reaction mixture was stirred under reflux for 5 hours . The solution was cooled and the product was precipitated by ether (100 ml) resulting in 2-Deoxy-2- [1- (4 , 4-dimethyl-2 , 6- dioxocyclohex-1-ylidene) ethylamino] -D-galactopyranose (21) (270 mg, 75%) .

Rf 0.37 (MeCN/H₂0 10:0.5);

FAB MS C₁₆H₂₅N0₇ (343.33) m/z (%) 366 [M+Na]⁺ (40), 344 [M+H]+ (100) , 327 (30) .

¹H NMR (D₂0) δ 5.34 (d, H-1&, Ji_/2= 3.54 Hz), 4.87 (d, H- l_β) , 4.28 (dd, H-2_α) , 4.17 (t, H-2_β) , 4.08 (d, H-4_α) , 4.03 (d, H-4_β) , 2.56 (s, 3H, CH₃ ) , 2.48, 2.44 (2s, 4H, 2 CH₂) , 1.03 (s, 6H, 2 CH₃ ) .

Example 20 Synthesis of Nde-protected aminosugars 2-Deoxy-2- [1 - (4 -ni tro-l , 3 -dioxoindan-2 -ylidene) -ethyl amino] -D-glucopyranose (22) Sodium (126 mg, 5.47 mmol) was added to abs. methanol (50 ml) and the reaction mixture was stirred for 5 min. D-glucosamine hydrochloride (1.18 g, 5.47 mmol) was added to the resulting clear solution and the reaction mixture was stirred at room temperature for another 5 min. 2- acetyl-4-nitroindane-l, 3-dion (1.91 g, 8.21 mmol) was added and the reaction mixture was stirred under reflux for 5 hours. The solution was cooled and the product was filtered off. The solid was washed with MeOH (10 ml), ether (50 ml) and dried, affording 2-Deoxy-2- [1- (4-nitro- 1,3- dioxoindan-2 -ylidene) ethylamino] -D-glucopyranose (22) (1.10 g, 55%) .

R_f 0.41 (MeCN/H₂0 10:0.5);

FAB MS C₁₇H₁₈N₂0₉ (394.32) m/z (%) 395 [M+H]⁺ (100).

NMR (D₂0) δ 7.75-7.40 (m, 3H, 3 Ar-H), 5.21 (d, H-l_α) , 3.95-3.25 (sugar 6H) , 3.18 (s, 3H, CH₃ ) .

Example 21 Synthesis of Nde - protected O-acetylated aminosugars 2-Deoxy-2- [1 - (4 -ni tro-l , 3 -dioxoindan-2 -ylidene) - ethylamino] -3 , 4 , 6-tri -O-acetyl -α-D- glucopyranose (23)

A mixture of 2-Deoxy-2- [1- (4-nitro-l, 3- dioxoindan-2 -ylidene) ethylamino] -D-glucopyranose (100 mg, 0.23 mmol), pyridine (2 ml) and acetic anhydride (3 ml) stirred at room temperature overnight. The reaction mixture was evaporated, and the residue was purified by chromatography using CHCl3/EtO c 10:4 as the mobile phase to give 2-Deoxy-2- [1- (4-nitro-l, 3 -dioxoindan-2 -ylidene) - ethylamino] -3,4, 6-tri-O-acetyl-α-D-glucopyranose (23) (165 mg, 79%) .

FAB MS C₂5H₂₆N₂0₁₃ (562.48) m/z (%) 585 [M+Na]⁺ (40), 563 [M+H]⁺ (100) , 503 (45) .

NMR (CDC1₃) δ 11.00, 10.90 (2d, IH, NH_{E Z}), 7.95-7.68

(m, 3H, 3 Ar-H), 6.25, 6.24 (2d, IH, H-1_E#Z), 5.43 (t, IH, H-3), 5.18 (t, IH, H-4), 2.68 (s, 3H, CH3 ) , 2.38, 2.07, 2.04, 2.00 (4s, 12H, 4 AcO). Example 22 Synthesis of Dde-protected deoxyaminosugars with furanose ring 3 ' -deoxy-3 ' - [1 - (4 , 4 -dimethyl -2 , 6 -dioxocyclohex-1 -ylidene) - ethylamino] -thymidine (24) 3 ' -Deoxy-3 ' -azido-thymidine (200 mg, 0.75 mmol) was dissolved in MeOH (25 ml) and Pd/C (40 mg) was added. The suspension was stirred over a constant stream of H₂ overnight . The reaction mixture was filtered, and the filtrate was concentrated. The residue was taken up in abs. EtOH (5 ml), N,N-diisopropylethylamine (0.1 ml) and 2-acetyldimedone (204 mg, 1.12 mmol) were added and the solution was refluxed for 5 h. The reaction mixture was cooled to room temperature and the product was precipitated by adding ether (50 ml) giving 3 ' -deoxy-3 ' - [1- (4, 4- dimethyl-2 , 6-dioxocyclohex-l-ylidene) -ethylamino] -thymidine (24) (200 mg, 66%) .

R_f 0.45 (CH₂Cl₂/EtOAc/MeOH 10:7:3);

FAB MS C₂₀H₂₇N₃θ4 (405.45) m/z (%) 428 [M+Na]⁺ (55), 406 [M+H]⁺ (100) .

^XH NMR (CDC1₃) δ 13.79 (d, IH, NH) , 7.55 (s, IH, H-6), 6.13 (m, IH, H-l'), 4.70 (m, IH, H-5'), 4.04 (m, IH, H-3 ' ) , 3.96 (dd, IH, H-5'_a), 3.72 (dd, IH, H-5'_b), 2.55 (s, 3H, CH3), 2.42 (m, IH, H-2'_a), 2.32 (s, 4H, 2 CH₂ ) , 1.80 (s, 3H, CH₃), 0.96 (s, 6H, 2 CH3 ) .

Example 23 Synthesis of Dde-protected aminosugar containing oligosaccharides

4 -0- (2, 3 , 4 , 6-tetra-O-acetyl - -D-galactopyranosyl ) -2, 3 , 6- tri -O-acetyl -N- [1 - (4 , 4 -dimethyl -2 , 6-dioxocyclohex-l - ylidene) ethyl] - -D-glucopyranosyl amine (27)

A mixture of β-lactose octaacetate (203 mg, 0.3 mmol), trimethylsilyl azide (41 mg, 0.35 mmol), and SnCl4 (40 mg,0.15 mmol) in CH₂C1₂ (1.5 ml) was stirred overnight at room temperature. The solution was diluted with CH₂C1₂ (20 ml) and washed twice with 1 M potassium fluoride solution (5 ml) , water (5 ml) and evaporated affording 4-0- (2,3,4, 6-tetra-O-acetyl-α-D-galacto- pyranosyl) -2 , 3 , 6-tri-O-acetyl-β-D-glucopyranosyl azide (25) (178 mg 90%) .^"

Rf 0.38 (hexane/EtOAc 1:1);

FAB MS C₂₆H3₅N₃0₁₇ (661.56) m/z (%) 684 [M+Na]⁺ (70), 662 [M+H]⁺ (20) , 331 (100) .

3-H NMR (CDC1₃) δ 5.35 (d, IH, H-4 ' ) , 4.95 (d, IH, H-l', J₁₍₂=3.63 Hz), 4.61 (d, IH, H-l, J_1/2=9.13 Hz), 2.14, 2.13, 2.07, 2.06, 2.04, 1.96 (6s, 21H, 7 AcO).

4-0- (2,3,4, 6-tetra-O-acetyl-α-D-galacto- pyranosyl) -2,3, 6-tri-O-acetyl-β-D-glucopyranosyl azide (178 mg, 0.26 mmol) was dissolved in MeOH (5 ml) and hydrogenated over Pd/C (10%) (10 mg) for 5 h. The suspension was filtered, and the filtrate was evaporated to give 4-0- (2,3,4, 6-tetra-O-acetyl-α-D-galacto-pyranosyl ) - 2 , 3 , 6-tri-O-acetyl-β-D-glucopyranosyl amine (26) (157 mg, 92%) .

Rf 0.41 (EtOAc) ;

FAB MS C₂6H₃₇N0₁₇ (635.56) m/z (%) 658 [M+Na]⁺ (35), 636 [M+H]⁺ (40) , 331 (100) .

NMR (CDCI3) δ 5.35 (d, IH, H-4'), 2.15, 2.12, 2.07, 2.06, 2.04, 2.03, 1.96 (7s, 21H, 7 AcO).

A mixture of 4-0- (2 , 3 , 4, 6-tetra-O-acetyl-α-D- galactopyranosyl) -2,3, 6-tri-O-acetyl-β-D-glucopyranosyl amine (157 mg, 0.24 mmol) and 2-acetyldimedone (81 mg, 0.45 mmol) in MeOH (5 ml) was refluxed for 5 h. The reaction mixture was evaporated, and the residue was purified by chromatography using CHCl3/EtOAc 1:1 as the mobile phase, to give 4-0- (2 , 3 , 4, 6-tetra-O- acetyl-α-D-galactopyranosyl) 2,3, 6-tri-O-acetyl-N- [1- (4, 4-dimethyl-2 , 6-dioxocyclohex-l- ylidene) ethyl] -β-D-glucopyranosyl amine (27) (106 mg, 54%)

Rf 0.39 (CHCl₃/EtOAc 1:1);

FAB MS C₃₆H49N0₁₉ (799.75) m/z (%) 822 [M+Na]⁺ (50), 800 [M+H]⁺ (100) .

iH NMR (CDC1₃) δ 13.56 (d, IH, NH) , 5.35 (d, IH, H-l', J₁₍ =3.13 Hz), 2.60 (s, 3H, CH₃ ) , 2.36 (s, 4H, 2 CH₂ ) , 2.15, 2.12, 2.07, 2.06, 2.04, 2.03, 1.96 (7s, 21H, 7 AcO), 1.02 (s, 6H, 2 CH₃) .

Example 24 Synthesis of 2-Acetyl-4-nitroindan-l, 3-dione 2 -Acetyl -4 -nitroindan-1 , 3-dione

A mixture of 3-nitrophthalic anydride (12 g, 60 mmol), anhydrous pyridine (25 ml), piperidine (0.2 ml) and 2 , 4-pentanedione (6.25 g, 60 mmol) was stirred at 40°C for 6 h. The reaction mixture was cooled to 0°C and the crystalline mass was collected at the pump, washed with ether, and dried to give the yellow pyridinium salt. The salt was treated with 6 M HC1 (100 ml) and the solid was filtered off. The product was crystallised from isopropanol to afford 2-Acetyl-4- nitroindan-1, 3-dione (8.74g, 79%) .

Rf 0.44 (EtOAc/AcOH 100:0.2) ;

FAB MS C_nH₇N0₅ (233.17) m/z (%) 256 [M+Na]+ (20) , 234 [M+H]+ (100) .

!H NMR (CDCI3) δ 8.09 -7.83 ( , 3H, 3 Ar- H_{(E; Z)}) , 2.62, 2.60 (2s, 3H, CH₃ (E,Z) ) • It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this invention.

References cited herein are listed on the following pages, and are incorporated by this reference.

REFERENCES

Boullanger, P., Banoub, J. and Descotes, G. Can. J. Chem. , 1987 65 1343.

Bovin, N.V., Zurabyan, S.E. and Khorlin, A.Y. Carbohydr. Chem., 1986 5_ 215.

Buskas, T. , Garegg, P.J., Konvadsson, P. and Maloisel, J.L.

Tetrahedron Asymmetry, 1994 5_ 2187.

Bycroft, B.W., Chan, W.C., Chhabra, S.R. and Hone, N.D. J. Chem. Soc. Chem. Commun., 1993 778.

Castro-Palomino, J.C. and Schmidt, R.R. Tetrahedron Lett., 1995 3_6 5343.

Chan, W.C., Bycroft, B.W., Evans, D.J. and White, P.D. J. Chem. Soc. Chem. Commun., 1995 2209.

Dasgupta, F. and Garegg, P.J.

J. Chem. Soc. Chem. Commun., 1989 1640

Fields, G.B. and Noble, R.L.

Int. J. Peptide Protein Res., 1990 3_5 161.

Griffith, D.A. and Danishefsky, S.J. J. Am. Chem. Soc, 1990 112 5811.

Hayakawa, Y., Kato, H., Uchiyama, M. , Kajino, H. and

Noyori, R.

J. Org. Chem., 1986 5_1 2400.

Imoto, M., Yoshimura, H., Shimamoto, T. , Sakahuchi , N. , Kusumoto, S. and Shiba, T. Bull. Chem. Soc. Jpn., 1987 60 2205. Kaifu, R. and Osawa, T. Carbohydr. Res., 1977 5_8 235.

Lemieux, R.V. , Abbas, S.Z. and Chung, B.Y. Can. J. Chem., 1982 60 58.

Lemieux, R.V. and Ratcliffe, R.T. Can. J. Chem., 1979 57 1244.

Mootoo, D.R. and Fraser-Reid. B. Tetrahedron Lett., 1989 3_0 2363.

Palsen, H. Angew. Chem. Int. Ed. Engl . , 1982 2JL 155.

Paulsen, H. and Stenzel, W., Chem. Ber., 1978 111 2334 & 2348.

Sasaki, T. , Minamoto, K. and Itoh, H. J. Org. Chem., 1978 _43 2320.

Schmidt, R.R. and Kinzy, W

Adv. Carbohydr. Chem. Biochem. , 1994 5_0 21-121.

Shapiro, D., Acher, A.J. and Rachaman, E.S. J. Org. Chem., 1967 3J2 3767.

Tailler, D., Jacquiaet, J.C, Noirot, A.M. and Bean, J.M.

J. Chem. Soc, Perkin. Trans. I, 1992 3163.

Toyokuni, T. and Singhal, A.K. Chem. Soc. Rev., 1995, 231.

Zurabyan, S.E., Antenenko, T.S. and Khorlin, A.Y. Carbohydr. Res., 1994 50 21-121

Claims

1. A compound containing a sugar carrying one or more primary amine groups protected with a 2-substituted- 1,3-dioxo compound of General Formula I or General Formula II:

II

in which

R¹ and R² may be the same or different, and is each hydrogen or C╧ç_ alkyl,

R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group, and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

2. A compound according to Claim 1, in which the protecting group is of General Formula I and R¹ and R² are both methyl .

3. A compound according to Claim 1, selected from the group consisting of Compounds 1 to 23 as described in Table 1, Compound 24 as described in Table 2 and compounds 25 to 27 as described in Table 3.

4. A reagent for solution phase synthesis of sugar- containing compounds, comprising a cyclic 2-substituted- 1,3-dioxo compound of General Formula I or General Formula II

II

in which

5. A reagent according to Claim 4 in which the protecting group is of General Formula I and both R¹ and R² are methyl .

6. A linker-saccharide complex, comprising a linker group and a saccharide compound comprising a protecting group of General Formula I or General Formula II

II

in which

R¹ and R² may be the same or different, and is each hydrogen or C╬╣_₄ alkyl, R' is an amino sugar, a glycosylamine, or an oligosaccharide comprising at least one aminosugar or one glycosylamine unit, in which the sugar is coupled via an amino group , and R" is alkyl, substituted alkyl, aryl, substituted aryl, cycloalkyl or substituted cycloalkyl.

7. A resin-linker-saccharide support for solid phase oligosaccharide synthesis, comprising a linker group, a resin, and a saccharide compound comprising a protecting group of General Formula I or General Formula II

II

in which

R¹ and R² may be the same or different, and is each hydrogen or C╬╣_ alkyl,

8. A method of solution phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a linker- saccharide complex as defined in Claim 6.

9. A method according to Claim 8 for synthesis of aminoglycoside compounds.

10. A method of solid-phase synthesis of oligosaccharides, comprising the step of sequentially linking mono- or oligosaccharide groups to a resin-linker- sugar support as defined in Claim 7.

11. A method according to any one of Claims 8 to 10 for combinatorial synthesis.

12. A kit for solid-phase synthesis or combinatorial synthesis of oligosaccharides, comprising a linker- saccharide complex according to Claim 6 or a resin-linker- saccharide support according to Claim 7, and optionally also comprising one or more further reagents such as partially or differentially activated, fully protected saccharides, protecting agents, deprotecting agents, resins and/or solvents suitable for solid phase or combinatorial synthesis .